It has been reported that PIWI-interacting RNAs (piRNAs) play critical roles in activating invasion and metastasis, evading growth suppressors, and sustaining proliferative signaling of cancer and can be regarded as a novel biomarker candidate. Thus, it is necessary to develop an effective method for imaging and regulating cancer-related piRNAs to diagnose and treat cancers. Herein, we designed aptamer-functionalized activatable DNA tetrahedron nanoprobes (apt-ADTNs) to image and regulate endogenous piRNAs in cancer cells. As proof of concept, overexpressed piRNA-36026 in MCF-7 cells was used for this study. In brief, aptamer AS1411 and piRNA-36026 antisequence with Cy5 fluorescent dye are appended from the DNA tetrahedron; then, a short oligonucleotide with black hole quencher 2 (Q-oligo) is complementary with piRNA-36026 antisequence to quench the fluorescence of Cy5. The apt-ADTNs can recognize the MCF-7 cells through aptamer AS1411, and then enter the cells. Q-oligo is detached from the apt-ADTNs because of the binding between apt-ADTNs and piRNA-36026, leading to the recovery of the Cy5 fluorescence signal. Meanwhile, the hybridization of apt-ADTNs and piRNA-36026 results in down-regulating of dissociative piRNA-36026 in cytoplasm and the subsequent apoptosis of MCF-7 cells. As the achievement of synchronously imaging and regulating piRNA-36026 in MCF-7 cells, we believe that this design holds great promise in application of diagnosis and therapy for cancer.